Treatment of bleeding with modified tissue factor in combination with FVII

ABSTRACT

It has been discovered that it is possible to administer truncated tissue factor, not having the transmembrane region (tTF) in combination with factor VIIa (FVIIa) to treat bleeding disorders such as those resulting from hemophilia or cirrhosis of the liver. Preferably, the tTF is administered to produce up to 10 μg tTF/ml of plasma. The FVIIa is administered to produce levels of between 40 ng FVIIa/ml and 4 μg FVIIa/ml of plasma. The effective dosages of both tTF and FVIIa are significantly and surprisingly less than the administration of either alone to stop bleeding. Examples demonstrate safety and efficacy in normal and hemophilic dogs.

The United States government has certain rights in this invention byvirtue of a grant awarded by the National Institutes of Health.

This is a continuation of U.S. Ser. No. 07/882,202 entitled "Treatmentof Bleeding with Modified Tissue Factor in Combination with FVIIa" filedMay 13, 1992, now U.S. Pat. No. 5,374,617, by James H. Morrissey andPhilip C. Comp and a continuation-in-part of copending U.S. Ser. No.07/683,682 entitled "Quactitative clotting Assay for Activated FactorFVII" filed Apr. 10, 1991 by James H. Morrissey.

BACKGROUND OF THE INVENTION

Blood coagulation results from the production of thrombin, a proteolyticenzyme inducing platelet aggregation and cleaving fihrinogen to fibrin,which stabilizes the platelet plug. A number of proenzymes andprocofactors circulating in the blood interact in this process throughseveral stages during which they are sequentially or simultaneouslyconverted to the activated form, ultimately resulting in the activationof prothromhin to thromhin by activated Factor X (fXa) in the presenceof Factor Va, ionic calcium, and platelets.

Factor X can he activated by either of two pathways, termed theextrinsic and intrinsic pathways. The intrinsic pathway, orsurface-mediated activation pathway, consists of a series of reactionswhere a protein precursor is cleaved to form an active protease,beginning with activation of Factor XII to Factor XIIa, which convertsFactor XI to Factor XIa, which, in the presence of calcium, convertsFactor IX to Factor IXa. Factors IX and X can also he activated via theextrinsic pathway by tissue factor (TF) in combination with activatedFactor VII (Factor VIIa; FVIIa). Activated Factor IX, in the presence ofcalcium, phospholipid (platelets), and Factor VIIIa, activates Factor Xto Factor Xa.

Physiologically, the major pathway involved in coagulation is believedto be the extrinsic pathway, an essential step of which is tissuefactor-mediated activation of Factor VII to Factor VIIa. Tissue factoris an integral membrane glycoprotein having a protein and a phospholipidcomponent. It has been isolated from a variety of tissues and speciesand reported to have a molecular mass of between 42,000 and 53,000. DNAencoding tissue factor and methods for expression of the protein havenow been reported, for example, in European Patent Application 0 278 776by Genentech, Inc. and by J. H. Morrissey, et al. Cell 50:129-135(1987).

The complex of Factor VIIa and its essential cofactor, TF, is the mostpotent known trigger of the clotting cascade. Factor VII is present inplasma at a concentration of 0.5 μg/ml plasma. In contrast, Factor VIIais present in plasma at trace levels of roughly 1 ng/ml. Accordingly,Factor VII is normally in considerable excess over Factor VIIa. FactorVIIa circulates with a relatively long half-life of about two hours inplasma. This is an unusual property among activated coagulation enzymes,which typically are inactivated very rapidly by various proteaseinhibitors in plasma.

Hemophilia A is characterized by the absence of active coagulationFactor VIII or the presence of inhibitors to Factor VIII. Hemophilia Bis characterized by the absence of active Factor IX. Both types ofhemophilia are associated with bleeding diatheses that can lead tocrippling injuries or death. Traditionally, patients with either type ofhemophilia were treated with infusion of plasma concentrates to stopbleeding episodes. The problems with the concentrates are many,especially infection, most recently with HIV. Highly purified FactorVIII or Factor IX have been introduced to obviate these problems, ashave methods of treating factor concentrates to inactivate viruses.However, some patients develop high-titer, inhibitory antibodies toFactor VIII. Therefore, such patients can no longer be treated withconventional Factor VIII replacement therapy.

As described by Hedner and Kisiel, J Clin Invest 71:1836-1841 (1983),purified naturally produced Factor VIIa can be administered tohemophilia A patients with high titers of antibodies against FactorVIII:C and restore hemostasis in these patients. As reported byBrinkhous, et al., Proc Natl Acad Sci USA 86:1382-1386 (1989),recombinant Factor VIIa (rFVIIa) can be administered to hemophilic andvon Willebrand disease dogs and stop bleeding in both hemophilic A and Bdogs, but not the von Willebrand disease dogs. Telgt, et al., ThrombosisRes 56:603-609 (1989), reported that, at high levels, rFVIIa wasbelieved to act by direct activation of Factor X, in the presence ofcalcium and phospholipid but in the absence of TF. Teitel, Thrombosisand Haemostasis 66:559-564 (1991), reported that the importantingredient in prothrombin complex concentrates for efficacy in treatinghemophilia is Factor VIIa.

Hedner, "Experiences with Recombinant Factor VIIa in Haemophiliacs" inBiotechnology of Plasma Proteins. Curr Stud Hematol Blood Transf,Lenfant, Mannucci, Sixma, eds., No. 58, 63-68 (1991), review the use ofprothrombin complex concentrates (effective in only 50 to 60% of thebleeds), as well as the use of plasma-derived (pFVIIa) and recombinantFactor VIIa (rFVIIa). Dosages of 10 to 15 μg/kg of pFVIIa were effectivein some hemophilia A patients. Safety studies in dogs and rabbitsindicated that the recombinant Factor VIIa was safe and efficacious atdosages of up to 150 μg/kg. A number of patients were also successfullytreated, using dosages of between 54 μg/kg and 90 μg/kg during surgeryand bleeding complications. Gringeri, et al., reported that treatment ofhemophiliacs with rFVIIa is not always effective, even at dosages of 75μg/kg at intervals of every two to three hours. The authors noted thatperhaps larger dosages, more frequent infusions, and/or the concomitantuse of antifibrinolytic medication might be required in such cases.rFVIIa is currently in clinical trials in the United States fortreatment of hemophilia, particularly hemophilia in patients withinhibitors who do not benefit from conventional Factor VIII or Factor IXreplacement therapy. Doses of rFVIIa currently employed are typically 45to 90 μg rFVIIa/kg body weight, and are repeated every two to fourhours. These doses are designed to achieve a level of circulating rFVIIaof approximately 4 μg/ml, extremely high compared to the normal plasmaconcentrations of FVII of approximately 0.5 μg/ml or FVIIa ofapproximately 1 ng/ml.

O'Brien, et al., J Clin Invest 82:206-211 (1988), reported that apo-TF,a delipidated preparation of the normally lipid-associated TFglycoprotein could be used to normalize bleeding in animals havingantibodies to Factor VIII. Since purified apo-TF is inactive unlessincorporated into a phospholipid membrane, the theoretical basis forinfusing apo-TF is the hypothesis that it would spontaneously andpreferentially incorporate into exposed membrane surfaces, particularlyinto damaged cells at the sites of injury. Subsequent studies haveindicated there are dangers associated with the use of purified apo-TFin the treatment of hemophilia. The apo-TF can spontaneously incorporateinto many types of lipid membranes and become active at sites whereclotting is not desired, resulting in thrombosis or disseminatedintravascular coagulation (DIC). Indeed, O'Brien, et al., reportedevidence of DIC in some animals receiving apo-TF, and Sakai and Kisiel,Thromb Res 60:213-222 (1990), recently demonstrated that apo-TF willspontaneously combine with plasma lipoproteins to form active TF. Thislatter phenomenon is cause for concern because of a number of studieswhich have demonstrated that intravenous administration of active TF isa potent inducer of DIC.

Recently, a soluble, truncated form of TF (tTF) has been reported whichretains some cofactor function towards Factor VIIa as measured in vitrousing purified proteins. However, this form of TF has been dismissed asan alternative to TF because it has almost no procoagulant activity whentested with normal plasma, as reported by Paborsky, et al., J Biol Chem266:21911-21916 (1991).

As described in U.S. Ser. No. 07/683,682 entitled "Quantitative ClottingAssay for Activated Factor VII" filed Apr. 10, 1991 by James H.Morrissey, the reason tTF was reported to lack procoagulant activity inthe prior art is because, although tTF retains cofactor function towardFactor VIIa, tTF had lost the ability to promote conversion of FactorVII to Factor VIIa. As a consequence, tTF can clot plasma only inconjunction with significantly elevated levels of Factor VIIa, ascompared to normal plasma, which contains only trace levels of FactorVIIa.

It is therefore an object of the present invention to provide a methodand composition for treatment of significant bleeding disorders, such ashemophilia, including those hemophiliacs with high titers of anti-FactorVIII antibodies.

It is a further object of the present invention to provide a method andcompositions for treatment of patients with bleeding disorders that arerelatively safe and can be produced in commercial quantities.

SUMMARY OF THE INVENTION

It has been discovered that it is possible to administer truncatedtissue factor (not having the transmembrane region) (tTF) in combinationwith Factor VIIa (FVIIa) to treat bleeding disorders such as, forexample, those associated with hemophilia or cirrhosis of the liver. ThetTF is administered to produce up to 10 μg tTF/ml of plasma. The FVIIais administered to produce levels of between 40 ng FVIIa/ml and 4 μgFactor VIIa/ml of plasma. The minimal effective dosages of both tTF andFVIIa are significantly and surprisingly less than the administration ofeither alone to stop bleeding.

Examples demonstrate safety and efficacy in normal and hemophilic dogs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a graph of dilute thromboplastin clotting time (seconds) forFactor VIII-deficient plasma when tTF and FVIIa (ng/ml) are added (darktriangles) or when FVIIa alone (ng/ml) are added (dark circles).

FIG. 1b is a graph of dilute thromboplastin clotting time (seconds) fornormal plasma when tTF and FVIIa (ng/ml) are added (dark triangles) orwhen FVIIa alone (ng/ml) are added (dark circles).

DETAILED DESCRIPTION OF THE INVENTION

Truncated tissue factor (tTF) is used as an adjuvant for Factor VIIa(FVIIa) therapy of excessive bleeding in patients such as hemophiliacs.The tTF is administered in combination with the FVIIa, or immediatelybefore or after the FVIIa. The minimal effective dosage of FVIIa issignificantly decreased by the tTF, while the thrombogenicity of themixture is reduced as compared to a mixture of FVIIa and TF in itsnative form.

The advantage, and necessity, of using a combination of FVIIa plus tTFover tTF alone is that tTF is active in promoting blood clotting only inthe presence of pre-formed FVIIa. The advantage of using the combinationof FVIIa plus tTF over FVIIa alone is that FVIIa is approximately22-fold more potent at clotting plasma in the presence of tTF than inthe absence of tTF. Furthermore, as with FVIIa alone, the complex of tTFplus FVIIa activates Factor X at markedly enhanced rates in the presenceof negatively charged phospholipids such as phosphatidylserine, eventhough tTF is not itself incorporated into lipid membranes. Normally,negatively charged phospholipids are sequestered into the inner leafletof the plasma membrane of cells, and thus are not exposed to the cellsurface unless the cells are damaged, or, in the case of platelets, theplatelets have undergone surface activation. Thus, negatively chargedphospholipids should be preferentially exposed on cell surfaces atanatomic sites that have experienced trauma sufficient to cause celllysis and/or platelet activation. This means that the complex of tTF andFVIIa is predicted from in vitro studies to be active in promoting bloodclotting preferentially at sites of injury, where it would be needed toconfer hemostasis. Furthermore, tTF, by itself or in complex with FVIIa,remains a soluble entity and does not become incorporated into lipidmembranes. This means that it should clear relatively rapidly from thecirculation, diminishing the risk of thrombosis. Because of theseproperties, the complex of tTF and FVIIa should not cause systemicactivation of the blood clotting system leading to DIC, as can injectionof wild-type TF. These in vitro results are consistent with results fromin vivo studies in normal and hemophiliac dogs described below.

The combination of tTF plus FVIIa should be useful for treatment ofhemophilia A, hemophilia B, congenital or acquired deficiencies in anyother blood coagulation factor, or platelet defects. Other patients thatcan be treated with the combination include patients suffering fromsevere trauma, postoperative bleeding or those with cirrhosis.

Truncated Tissue Factor

As used herein, "truncated tissue factor" is a soluble tissue factorhaving only the extracellular domains, which is not bound to aphospholipid membrane surface, and therefore does not support conversionof FVII to FVIIa, as described in U.S. Ser. No. 07/683,682 filed Apr.10, 1991, corresponding to International Application PCT/US92/02898filed Apr. 9, 1992, the teachings of which are incorporated herein. Inthe preferred embodiment, truncated tissue factor is a recombinantprotein produced in vitro in cell culture using a mammalian cell linesuch as CHO-K1 cells, American Type Culture Collection CCL 61. Thesecells are stably transfected with a mutant form of the human tissuefactor cDNA carried in a commercially available expression vector, andsecrete a form of tissue factor consisting only of amino acids 1-219(numbered according to Morrissey, et al., Cell 50:129-135 (1987), theteachings of which are incorporated herein).

The recombinant, truncated tissue factor is purified from the culturemedium using an immobilized monoclonal antibody to human tissue factor,such as TF9-5B7, described in Morrissey, et al., Thromb Res 52:247-261(1988). Hybridomas for production of monoclonal antibodies can bepropagated by ascites growth and the monoclonal antibodies (MAbs)purified from ascites fluid using the BioRad MAPS II system for mAbpurification, as described by Morrissey, et al., Thromb Res 52:247-261(1988).

The TF9-5B7 is coupled to Affigel™ beads. Detergent is not used duringthe purification of TF₂₁₉. After removal of cellular debris bycentrifugation, the culture medium is made 25 mM in Tris.HCl (pH 7.4),10 mM in sodium EDTA (pH 7.4), and 0.1% in sodium azide by the additionof concentrated stock solutions. In order to remove proteins that bindto agarose beads non-specifically, the culture medium is gently agitatedfor 4 hr at 4° C. with AffiGel™-10 beads that had previously beenblocked chemically with glycine ethyl ester (GEE-AffiGel). TheGEE-AffiGel beads are removed by filtration through a sintered glassfunnel, and the supernatant is agitated overnight at 4° C. with the MAbTF9-5B7 coupled to AffiGel beads (typically 2 ml of beads). TheTF9-5B7-AffiGel beads are collected on a sintered glass funnel, and thebeads are washed on the funnel with 100 ml of TBS-EDTA (TBS=100 mM NaCl,50 mM Tris.HCl (pH 7.4) and 0.02% sodium azide; TBS-EDTA=TBS with 10 mMEDTA included). The beads are then transferred to a chromatographycolumn and washed with 40 ml TBS followed by 40 ml of a solutionconsisting of 1 M NaCl, 10 mM Tris. HCl (pH 7.4), and 0.02% sodiumazide. Truncated TF is eluted from the beads using 100 mM glycine. HCl(pH 2.5), with 1 ml fractions being collected into tubes containing 57μl 1 M Tris base (to immediately neutralize the acidic glycine buffer).Fractions containing protein are detected using the BCA protein assay(Pierce Chemical Co., Rockford, ILL.), pooled, dialyzed against TBS, andthen stored at -70° C. Protein concentrations are determined usingstandard methods such as the BCA assay (Pierce Chemical Co.) based on abovine serum albumin standard of known concentration.

For production of recombinant truncated TF, cells are typically grown toconfluence in roller bottles in Dulbecco's Modified Eagle's Medium(DMEM) supplemented with 10% iron-supplemented calf serum (HyCloneLaboratories, Logan, Utah). Upon reaching confluence, the cultures areshifted to DMEM containing 2% serum, and culture medium is collectedevery four days thereafter.

The nucleotide and amino acid sequence of truncated tissue factor (tTF)is shown below as SEQ ID NO:1 and SEQ ID NO:2, respectively. Thetruncated tissue factor protein lacks the predicted transmembrane andcytoplasmic domains of tissue factor. This version of the proteinretains cofactor activity, as reported by Ruf, et al., Thromb Haemost62:347 (abstract) (1989) and Ruf, et al., J Biol Chem 266:2158-2166(1991). This truncated form of tissue factor fails to support conversionof Factor VII to Factor VIIa, allowing it to be used in a specificclotting assay for Factor VIIa free from interference by Factor VII.

Since the vast majority of FVII in plasma is in the inactive, zymogenform, and since tTF is selectively deficient in promoting conversion ofFactor VII to Factor VIIa, tTF has extremely little procoagulantactivity towards normal plasma. Since tTF lacks the membrane-anchoringdomain of the protein, it lacks the ability to become incorporated intoexposed phospholipid surfaces as wild-type apo-TF can. Although thedeficiency of tTF in the conversion of FVII to FVIIa was not previouslyappreciated, its extremely low procoagulant activity when tested withnormal plasma has made it appear to be unacceptable as a therapeuticagent to control bleeding in hemophilia.

The essential difference between truncated tissue factor and wild-typetissue factor is that truncated tissue factor is no longer tethered tothe phospholipid membrane surface. It is therefore expected that othermethods for preparing truncated tissue factor can be used to generate anessentially equivalent soluble form of tissue factor that retains FVIIacofactor activity while no longer stimulating conversion of Factor VIIto Factor VIIa. Methods include chemical and/or enzymatic cleavage ofwild-type tissue factor to separate the predicted extracellular domainfrom the transmembrane region. Recombinant human TF is available fromCalbiochem Corporation. Precise positioning of the stop codon followingamino acid 219 is believed to not be essential to make functionaltruncated TF, and other placements of a stop codon near amino acid 219are predicted to yield an essentially equivalent product with respect toits ability in conjugation with FVIIa to serve as a treatment forbleeding disorders.

Activated Factor VII

Factor VII can be prepared as described by Fair, Blood 62:784-791(1983). The coding portion of the human Factor VII cDNA sequencereported by Hagen et al., Proc Natl Acad Sci USA 83:2412-2416 (1986) isshown as SEQ ID NO:3, along with the translated amino acid sequence, SEQID NO:4. The amino acid sequence from 1 to 60 corresponds to thepre-pro/leader sequence that is removed by the cell prior to secretion.The mature FVII polypeptide chain consists of amino acids 61 to 466.FVII is converted to FVIIa by cleavage of a single peptide bond betweenarginine-212 and isoleucine-213.

FVII can be converted in vitro to FVIIa by incubation of the purifiedprotein with Factor Xa immobilized on Affi-Gel™ 15 beads (Bio-Rad).Conversion can be monitored by SDS-polyacrylamide gel electrophoresis ofreduced samples. Free Factor Xa in the FVIIa preparation can be detectedwith the chromogenic substrate methoxycarbonyl-D-cyclohexylglycyl-glycyl-arginine-p-nitroanilide acetate (Spectrozyme™FXa, American Diagnostica, Greenwich, Conn.) at 0.2 mM finalconcentration in the presence of 50 mM EDTA.

Recombinant FVIIa can also be purchased from Novo Biolabs (Danbury,Conn.).

Administration of the combination of the tTF and FVIIa

The tTF and FVIIa are administered by infusion in the preferredembodiment, using a pharmaceutically acceptable carrier such as salineor buffered saline. The tTF and FVIIa can also be administered topicallyeither by direct application using a conventional topical base such aspetrolatum or a water based gel, or as an aerosol spray.

The tTF is administered in a dosage effective to produce in the plasmaan effective level of between 100 ng/ml and 50 μg/ml, or a preferredlevel of between 1 μg/ml and 10 μg/ml or 60 to 600 μg/kg body weight,when administered systemically; or an effective level of between 10μg/ml and 50 μg/ml, or a preferred level of between 10 μg/ml and 50μg/ml, when administered topically.

The FVIIa is administered in a dosage effective to produce in the plasmaan effective level of between 20 ng/ml and 10 μg/ml (1.2 to 600 μg/kg),or a preferred level of between 40 ng/ml and 4 μg/ml (2.4 to 240 μg/kg),or a level of between 1 μg/ml and 10 μg/ml FVIIa when administeredtopically.

In general, one would administer tTF and FVIIa to produce levels of upto 10 μg tTF/ml plasma and between 40 ng and 4 μg FVIIa/ml plasma. Forhemophilic patients, one would administer tTF and FVIIa to producelevels of up to 10 μg tTF and between 100 and 300 ng FVIIa/ml. Forpatients with cirrhosis, one would administer the same amount of tTF butup to 1 μg FVIIa/ml plasma.

Since tTF cannot stimulate conversion of FVII to FVIIa, it should onlybe functional in conjunction with elevated factor VIIa levels.Therefore, tTF is expected to be an effective hemostatic agent only inassociation with FVIIa therapy or in individuals who have elevated FVIIalevels for some other reason.

Other conditions that can be treated with this combination includesurgical bleeding from the microvasculature, bleeding at skin graftharvest sites, postoperative bleeding, including following orthopedicsurgery, brain surgery or brain trauma, bleeding secondary tothrombocytopenia, and platelet dysfunction.

EXAMPLE 1

Safety,of tTF and FVIIa in normal dogs.

Three beagle dogs were treated with either tTF or tTF plus FVIIa.

Dog #5533 was treated with tTF alone, in a dosage of 60 μg tTF/kg bodyWeight by intravenous bolus infusion. ELISA studies showed 1.23 μgtTF/ml in plasma at time zero. Half-life of tTF was 2.2 hr in this dog.

Dog #5534 was also treated with tTF alone, in a dosage of 41 μg tTF/kgbody weight by intravenous bolus infusion. Assuming a blood volume of 60ml/kg, this should have produced a level of 0.67 μg tTF/ml in the blood.ELISA studies showed 0.60 μg tTF/ml in plasma at time zero. Half-life oftTF was 1.3 hr in this dog.

Dog #5734 was treated with tTF in combination with FVIIa, in a dosage of41 μg tTF/kg body weight and 6.37 μg Novo recombinant factor VIIa/kgbody weight by intravenous bolus infusion. ELISA studies showed 0.51 μgtTF/ml in plasma at time zero. Half-life of tTF was biphasic (1 hrfollowed by 3.0 hr) in this dog.

The dogs all remained healthy and active. Blood tests of the clottingsystem indicated no significant decrease in fibrinogen levels orplatelet counts and no measurable increase in fibrin degradationproducts. Prothrombin times and APTT times were normal, as were whiteblood cell counts, and red blood cell counts. Therefore, administrationof either tTF alone, or tTF in conjunction with FVIIa, caused nomeasurable disseminated intravascular coagulation or other detectablecoagulopathy.

A complete necropsy was performed on the animal receiving truncatedtissue factor and factor VIIa. No evidence of thrombosis was found inveins, arteries or in the capillaries. There was no evidence on grossand microscopic examination of myocardial infarction or of cerebralinfarction (stroke).

EXAMPLE 2

In vitro correction of clotting time of hemophilic plasma with tTF incombination with FVIIa.

A modified prothrombin test (PT) was performed with dilutedthromboplastin (since hemophiliacs have normal PT's unless thethromboplastin is diluted) using either normal or congenital factor VIIIdeficient (Hemophilia A) plasma. Sigma Chemical Co. rabbit brainthromboplastin was diluted 1:500 with TBS/0.1% BSA/rabbit brain cephalin(Sigma Chemical Co., St. Louis, Mo.).

12×75 mm glass test tubes were pre-warmed in a 37° C. water bath.

Diluted thromboplastin was added (0.1 ml) and allowed to warm to 37° C.for more than two minutes.

Plasma sample (0.1 ml) was added and allowed to warm to exactly 30 sec.

Pre-warmed 25 mM CaCl.sub. 2 (at 37° C.) was added and the clotting timewas determined by the manual tilt-tube method.

The results are shown in FIG. 1a and 1b. FIG. 1a is a graph of dilutethromboplastin clotting time (seconds) for factor VIII-deficient plasmawhen tTF (1 μg/ml) and varying concentrations of FVIIa (ng/ml) are added(dark triangles) or when varying concentrations of FVIIa alone (ng/ml)are added (dark circles). FIG. 1b is a graph of dilute thromboplastinclotting time (seconds) for normal plasma when tTF (1 μg/ml) and varyingconcentrations FVIIa (ng/ml) are added (dark triangles) or when varyingconcentration of FVIIa alone (ng/ml) are added (dark circles). Theclotting time of the hemophilic plasma without any added FVIIa or tTFwas 88.5 sec which is indicated by the upper dotted horizontal line. Theclotting time of normal plasma without added FVIIa or tTF was 53,0 secwhich is indicated by the lower dotted horizontal line. Without addedtTF, the 88.5 sec clotting time of the hemophilic plasma was reduced tothat of normal plasma (53 sec) at 808 ng FVIIa/ml of plasma. With addedtTF (at 1 μg/ml), the clotting time of the hemophilic plasma was reducedto that of normal plasma at 36.8 ng FVIIa/ml plasma.

Therefore, in the presence of 1 μg/ml tTF in plasma, correction of theprolonged clotting time of hemophilic plasma was achieved at a level ofadded FVIIa that was 22-fold lower than in the absence of added tTF. Inaddition, tTF was not able to correct the prolonged clotting time ofhemophilic plasma in the absence of added FVIIa.

EXAMPLE 3

Efficacy of the combination of tTF and FVIIa in the treatment ofHemophilic Dogs.

As described by Brinkhous, et al., Proc Natl Acad Sci USA 82:8752-8756(1985), Graham, et al., J Exp Med 90:97-111 (1949), and Brinkhous, etal., Ann NY Acad Sci 370:191-204 (1981), a colony of hemophilic dogs hasbeen developed at the University of North Carolina at Chapel Hill. Dogshave hemophilia A. A test (secondary cuticle bleeding time) is used tomeasure bleeding tendency.

A modified toenail bleeding time (BT) is used to test the hemostaticeffectiveness of infused preparations. The paw of the front leg iswarmed by placing it in isotonic saline at 37° C. and a toenail istrimmed to expose only the distal matrix. The bleeding nail is placed inisotonic saline at 37° C. and the time until cessation of bleedingrecorded as the primary BT. At 2 to 4 hours, the site is shaved toremove the clot and as little nail matrix as possible. The paw is againplaced in saline at 37° C. A discrete stream of extruding blood isvisible. With hemophilic dogs in the absence of treatment, bleedingtypically continues for 30 min or longer. This time to cessation ofbleeding is recorded as a secondary BT. For normal dogs, the primary BTis two to five minutes and the secondary BT is less than five minutes.For hemophilic dogs the primary BT is similar to that of normal dogswhile secondary BT is greater than 15 minutes.

Dog #V02 was administered a low dose of FVIIa, 6 μg FVIIa/kg bodyweight, which was designed to provide only a weak hemostatic effect.After 15 min equilibrium, secondary cuticle bleeding time was measuredat 2 min 30 Sec, which spontaneously rebled for 5 min 15 sec. Rebleedingchallenge (wipe off clot with gauze): 9 min 20 sec bleeding time, whichagain spontaneously rebled for 15 min, at which time the nail wascauterized to prevent further bleeding. It was difficult to stopbleeding and oozing from the phlebotomy sites in this dog, whichcontinued after FVIIa administration.

The results indicate that this dosage of FVIIa had a very weakhemostatic effect.

The dog was then administered 42 μg tTF/kg body weight. After 15 minequilibration, secondary cuticle bleeding time was measured at 50 sec,which did not spontaneously rebleed. This was dramatically shorter thanwhen the animal had received FVIIa alone. Wiping the clot off with gauzeto provoke rebleeding caused rebleeding for only 35 sec, and nosubsequent rebleeding was observed. Oozing from two phlebotomy sites inforepaw veins also stopped following infusion of tTF.

The results indicated that tTF in combination with low dose FVIIa has anexcellent hemostatic effect.

The same dog was administered 42 μg tTF/kg body weight, without FVIIa,two days later (since the half-lives of both tTF and FVIIa are about 2hr, this was more than sufficient to ensure clearance of eithersubstance from the plasma). After 15 minutes equilibration, thesecondary cuticle bleeding time was greater than 15 minutes and had tobe stopped by cauterization.

The results indicated that tTF alone had no measurable hemostaticeffect.

The dog was then administered 6 μg FVIIa/kg body weight. After 15minutes equilibration, secondary cuticle bleeding time was measured at 3minutes 15 seconds, which did not spontaneously rebleed. Wiping the clotoff with gauze caused rebleeding for 3 minutes, and no subsequentrebleeding was observed.

The results again demonstrate that tTF in combination with low doseFVIIa has a very good hemostatic effect.

Modifications and variations of the present invention, a method andcompositions for the treatment of excessive bleeding, will be obviousfrom the foregoing detailed description and are intended to come withinthe scope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 4    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 795 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO     (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Homo sapiens    (F) TISSUE TYPE: Fibroblast    (ix) FEATURE:    (A) NAME/KEY: CDS    (B) LOCATION: 34..786    (ix) FEATURE:    (A) NAME/KEY: mat-peptide    (B) LOCATION: 130..786    (D) OTHER INFORMATION: /product="Truncated Tissue Factor"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    CGTTCCGCTCGATCTCGCCGCCAACTGGTAGACATGGAGACCCCTGCCTGGCCC54    MetGluThrProAlaTrpPro    32 -30    CGGGTCCCGCGCCCCGAGACCGCCGTCGCTCGGACGCTCCTGCTCGGC102    ArgValProArgProGluThrAlaValAlaArgThrLeuLeuLeuGly    25-20-15 -10    TGGGTCTTCGCCCAGGTGGCCGGCGCTTCAGGCACTACAAATACTGTG150    TrpValPheAlaGlnValAlaGlyAlaSerGlyThrThrAsnThrVal    51 5    GCAGCATATAATTTAACTTGGAAATCAACTAATTTCAAGACAATTTTG198    AlaAlaTyrAsnLeuThrTrpLysSerThrAsnPheLysThrIleLeu    1015 20    GAGTGGGAACCCAAACCCGTCAATCAAGTCTACACTGTTCAAATAAGC246    GluTrpGluProLysProValAsnGlnValTyrThrValGlnIleSer    2530 35    ACTAAGTCAGGAGATTGGAAAAGCAAATGCTTTTACACAACAGACACA294    ThrLysSerGlyAspTrpLysSerLysCysPheTyrThrThrAspThr    404550 55    GAGTGTGACCTCACCGACGAGATTGTGAAGGATGTGAAGCAGACGTAC342    GluCysAspLeuThrAspGluIleValLysAspValLysGlnThrTyr    6065 70    TTGGCACGGGTCTTCTCCTACCCGGCAGGGAATGTGGAGAGCACCGGT390    LeuAlaArgValPheSerTyrProAlaGlyAsnValGluSerThrGly    7580 85    TCTGCTGGGGAGCCTCTGTATGAGAACTCCCCAGAGTTCACACCTTAC438    SerAlaGlyGluProLeuTyrGluAsnSerProGluPheThrProTyr    9095 100    CTGGAGACAAACCTCGGACAGCCAACAATTCAGAGTTTTGAACAGGTG486    LeuGluThrAsnLeuGlyGlnProThrIleGlnSerPheGluGlnVal    105110 115    GGAACAAAAGTGAATGTGACCGTAGAAGATGAACGGACTTTAGTCAGA534    GlyThrLysValAsnValThrValGluAspGluArgThrLeuValArg    120125130 135    AGGAACAACACTTTCCTAAGCCTCCGGGATGTTTTTGGCAAGGACTTA582    ArgAsnAsnThrPheLeuSerLeuArgAspValPheGlyLysAspLeu    140145 150    ATTTATACACTTTATTATTGGAAATCTTCAAGTTCAGGAAAGAAAACA630    IleTyrThrLeuTyrTyrTrpLysSerSerSerSerGlyLysLysThr    155160 165    GCCAAAACAAACACTAATGAGTTTTTGATTGATGTGGATAAAGGAGAA678    AlaLysThrAsnThrAsnGluPheLeuIleAspValAspLysGlyGlu    170175 180    AACTACTGTTTCAGTGTTCAAGCAGTGATTCCCTCCCGAACAGTTAAC726    AsnTyrCysPheSerValGlnAlaValIleProSerArgThrValAsn    185190 195    CGGAAGAGTACAGACAGCCCGGTAGAGTGTATGGGCCAGGAGAAAGGG774    ArgLysSerThrAspSerProValGluCysMetGlyGlnGluLysGly    200205210 215    GAATTTAGAGAATAAGAATTC795    GluPheArgGlu    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 251 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii ) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    MetGluThrProAlaTrpProArgValProArgProGluThrAlaVal    32-30-25-20    AlaArgThrLeuLeuLeuGlyTrpValPheAlaGlnValAl aGlyAla    15-10-5    SerGlyThrThrAsnThrValAlaAlaTyrAsnLeuThrTrpLysSer    151015    ThrAsn PheLysThrIleLeuGluTrpGluProLysProValAsnGln    202530    ValTyrThrValGlnIleSerThrLysSerGlyAspTrpLysSerLys    35 4045    CysPheTyrThrThrAspThrGluCysAspLeuThrAspGluIleVal    505560    LysAspValLysGlnThrTyrLeuAlaArgValP heSerTyrProAla    65707580    GlyAsnValGluSerThrGlySerAlaGlyGluProLeuTyrGluAsn    8590 95    SerProGluPheThrProTyrLeuGluThrAsnLeuGlyGlnProThr    100105110    IleGlnSerPheGluGlnValGlyThrLysValAsnValThrVa lGlu    115120125    AspGluArgThrLeuValArgArgAsnAsnThrPheLeuSerLeuArg    130135140    AspValPheGlyLys AspLeuIleTyrThrLeuTyrTyrTrpLysSer    145150155160    SerSerSerGlyLysLysThrAlaLysThrAsnThrAsnGluPheLeu    165 170175    IleAspValAspLysGlyGluAsnTyrCysPheSerValGlnAlaVal    180185190    IleProSerArgThrValAsnArgL ysSerThrAspSerProValGlu    195200205    CysMetGlyGlnGluLysGlyGluPheArgGlu    210215    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 1440 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA    (iii) HYPOTHETICAL: NO    (iv) ANTI-SENSE: NO    (vi) ORIGINAL SOURCE:    (A) ORGANISM: Homo sapiens    (F) TISSUE TYPE: Blood    (ix) FEATURE:    (A) NAME/KEY: CDS     (B) LOCATION: 36..1433    (D) OTHER INFORMATION: /note="Coding portion of human    factor VII cDNA"    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    TCAACAGGCAGGGGCAGCACTGCAGAGATTTCATCATGGTCTCCCAGGCCCTC53    Met ValSerGlnAlaLeu    15    AGGCTCCTCTGCCTTCTGCTTGGGCTTCAGGGCTGCCTGGCTGCAGGC101    ArgLeuLeuCysLeuLeuLeuGlyLeuGln GlyCysLeuAlaAlaGly    101520    GGGGTCGCTAAGGCCTCAGGAGGAGAAACACGGGACATGCCGTGGAAG149    GlyValAlaLysAlaSerGlyGlyGluThr ArgAspMetProTrpLys    253035    CCGGGGCCTCACAGAGTCTTCGTAACCCAGGAGGAAGCCCACGGCGTC197    ProGlyProHisArgValPheValThrGlnGlu GluAlaHisGlyVal    404550    CTGCACCGGCGCCGGCGCGCCAACGCGTTCCTGGAGGAGCTGCGGCCG245    LeuHisArgArgArgArgAlaAsnAlaPheLeuGluGlu LeuArgPro    55606570    GGCTCCCTGGAGAGGGAGTGCAAGGAGGAGCAGTGCTCCTTCGAGGAG293    GlySerLeuGluArgGluCysLysGluGluGln CysSerPheGluGlu    758085    GCCCGGGAGATCTTCAAGGACGCGGAGAGGACGAAGCTGTTCTGGATT341    AlaArgGluIlePheLysAspAlaGluArg ThrLysLeuPheTrpIle    9095100    TCTTACAGTGATGGGGACCAGTGTGCCTCAAGTCCATGCCAGAATGGG389    SerTyrSerAspGlyAspGlnCysAlaSer SerProCysGlnAsnGly    105110115    GGCTCCTGCAAGGACCAGCTCCAGTCCTATATCTGCTTCTGCCTCCCT437    GlySerCysLysAspGlnLeuGlnSerTyrIle CysPheCysLeuPro    120125130    GCCTTCGAGGGCCGGAACTGTGAGACGCACAAGGATGACCAGCTGATC485    AlaPheGluGlyArgAsnCysGluThrHisLysAspAsp GlnLeuIle    135140145150    TGTGTGAACGAGAACGGCGGCTGTGAGCAGTACTGCAGTGACCACACG533    CysValAsnGluAsnGlyGlyCysGluGlnTyr CysSerAspHisThr    155160165    GGCACCAAGCGCTCCTGTCGGTGCCACGAGGGGTACTCTCTGCTGGCA581    GlyThrLysArgSerCysArgCysHisGlu GlyTyrSerLeuLeuAla    170175180    GACGGGGTGTCCTGCACACCCACAGTTGAATATCCATGTGGAAAAATA629    AspGlyValSerCysThrProThrValGlu TyrProCysGlyLysIle    185190195    CCTATTCTAGAAAAAAGAAATGCCAGCAAACCCCAAGGCCGAATTGTG677    ProIleLeuGluLysArgAsnAlaSerLysPro GlnGlyArgIleVal    200205210    GGGGGCAAGGTGTGCCCCAAAGGGGAGTGTCCATGGCAGGTCCTGTTG725    GlyGlyLysValCysProLysGlyGluCysProTrpGln ValLeuLeu    215220225230    TTGGTGAATGGAGCTCAGTTGTGTGGGGGGACCCTGATCAACACCATC773    LeuValAsnGlyAlaGlnLeuCysGlyGlyThr LeuIleAsnThrIle    235240245    TGGGTGGTCTCCGCGGCCCACTGTTTCGACAAAATCAAGAACTGGAGG821    TrpValValSerAlaAlaHisCysPheAsp LysIleLysAsnTrpArg    250255260    AACCTGATCGCGGTGCTGGGCGAGCACGACCTCAGCGAGCACGACGGG869    AsnLeuIleAlaValLeuGlyGluHisAsp LeuSerGluHisAspGly    265270275    GATGAGCAGAGCCGGCGGGTGGCGCAGGTCATCATCCCCAGCACGTAC917    AspGluGlnSerArgArgValAlaGlnValIle IleProSerThrTyr    280285290    GTCCCGGGCACCACCAACCACGACATCGCGCTGCTCCGCCTGCACCAG965    ValProGlyThrThrAsnHisAspIleAlaLeuLeuArg LeuHisGln    295300305310    CCCGTGGTCCTCACTGACCATGTGGTGCCCCTCTGCCTGCCCGAACGG1013    ProValValLeuThrAspHisValValProLeu CysLeuProGluArg    315320325    ACGTTCTCTGAGAGGACGCTGGCCTTCGTGCGCTTCTCATTGGTCAGC1061    ThrPheSerGluArgThrLeuAlaPheVal ArgPheSerLeuValSer    330335340    GGCTGGGGCCAGCTGCTGGACCGTGGCGCCACGGCCCTGGAGCTCATG1109    GlyTrpGlyGlnLeuLeuAspArgGlyAla ThrAlaLeuGluLeuMet    345350355    GTGCTCAACGTGCCCCGGCTGATGACCCAGGACTGCCTGCAGCAGTCA1157    ValLeuAsnValProArgLeuMetThrGlnAsp CysLeuGlnGlnSer    360365370    CGGAAGGTGGGAGACTCCCCAAATATCACGGAGTACATGTTCTGTGCC1205    ArgLysValGlyAspSerProAsnIleThrGluTyrMet PheCysAla    375380385390    GGCTACTCGGATGGCAGCAAGGACTCCTGCAAGGGGGACAGTGGAGGC1253    GlyTyrSerAspGlySerLysAspSerCysLys GlyAspSerGlyGly    395400405    CCACATGCCACCCACTACCGGGGCACGTGGTACCTGACGGGCATCGTC1301    ProHisAlaThrHisTyrArgGlyThrTrp TyrLeuThrGlyIleVal    410415420    AGCTGGGGCCAGGGCTGCGCAACCGTGGGCCACTTTGGGGTGTACACC1349    SerTrpGlyGlnGlyCysAlaThrValGly HisPheGlyValTyrThr    425430435    AGGGTCTCCCAGTACATCGAGTGGCTGCAAAAGCTCATGCGCTCAGAG1397    ArgValSerGlnTyrIleGluTrpLeuGlnLys LeuMetArgSerGlu    440445450    CCACGCCCAGGAGTCCTCCTGCGAGCCCCATTTCCCTAGCCCA1440    ProArgProGlyValLeuLeuArgAlaProPhePro    4 55460465    (2) INFORMATION FOR SEQ ID NO:4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 466 amino acids    (B) TYPE: amino acid    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    MetValSerGlnAlaLeuArgLeuLeuCy sLeuLeuLeuGlyLeuGln    151015    GlyCysLeuAlaAlaGlyGlyValAlaLysAlaSerGlyGlyGluThr    2025 30    ArgAspMetProTrpLysProGlyProHisArgValPheValThrGln    354045    GluGluAlaHisGlyValLeuHisArgArgArgArgAlaAsnAlaPhe    505560    LeuGluGluLeuArgProGlySerLeuGluArgGluCysLysGluGlu    65707580    GlnCysSerP heGluGluAlaArgGluIlePheLysAspAlaGluArg    859095    ThrLysLeuPheTrpIleSerTyrSerAspGlyAspGlnCysAlaSer    100 105110    SerProCysGlnAsnGlyGlySerCysLysAspGlnLeuGlnSerTyr    115120125    IleCysPheCysLeuProAlaPheGluGl yArgAsnCysGluThrHis    130135140    LysAspAspGlnLeuIleCysValAsnGluAsnGlyGlyCysGluGln    145150155 160    TyrCysSerAspHisThrGlyThrLysArgSerCysArgCysHisGlu    165170175    GlyTyrSerLeuLeuAlaAspGlyValSerCysThrProThrVal Glu    180185190    TyrProCysGlyLysIleProIleLeuGluLysArgAsnAlaSerLys    195200205    ProGlnGlyA rgIleValGlyGlyLysValCysProLysGlyGluCys    210215220    ProTrpGlnValLeuLeuLeuValAsnGlyAlaGlnLeuCysGlyGly    225230 235240    ThrLeuIleAsnThrIleTrpValValSerAlaAlaHisCysPheAsp    245250255    LysIleLysAsnTrpArgAsnLeuIl eAlaValLeuGlyGluHisAsp    260265270    LeuSerGluHisAspGlyAspGluGlnSerArgArgValAlaGlnVal    275280 285    IleIleProSerThrTyrValProGlyThrThrAsnHisAspIleAla    290295300    LeuLeuArgLeuHisGlnProValValLeuThrAspHisValValPro    305 310315320    LeuCysLeuProGluArgThrPheSerGluArgThrLeuAlaPheVal    325330335    ArgPheS erLeuValSerGlyTrpGlyGlnLeuLeuAspArgGlyAla    340345350    ThrAlaLeuGluLeuMetValLeuAsnValProArgLeuMetThrGln    355 360365    AspCysLeuGlnGlnSerArgLysValGlyAspSerProAsnIleThr    370375380    GluTyrMetPheCysAlaGlyTyrSerAspGlySe rLysAspSerCys    385390395400    LysGlyAspSerGlyGlyProHisAlaThrHisTyrArgGlyThrTrp    405410 415    TyrLeuThrGlyIleValSerTrpGlyGlnGlyCysAlaThrValGly    420425430    HisPheGlyValTyrThrArgValSerGlnTyrIleGluTrpLeu Gln    435440445    LysLeuMetArgSerGluProArgProGlyValLeuLeuArgAlaPro    450455460    PhePro    465

We claim:
 1. A method for treating a patient for excessive bleedingoccurring at a site, comprising the step of:simultaneously administeringan effective amount of a truncated tissue factor protein and aneffective amount of Factor VIIa to a patient to effect clotting of saidpatient's blood at said site and subsequent reduction of said excessivebleeding, said truncated tissue factor protein having an amino acidsequence beginning at position 1 and terminating near position 219 ofSEQ ID NO:2, said truncated tissue factor protein differing from that ofnative tissue factor in that transmembrane amino acids selected from themembrane spanning domain of native tissue factor which would besufficient to bind said truncated tissue factor protein to phospholipidmembranes are lacking from said truncated tissue factor protein so thatsaid truncated tissue factor protein does not bind to phospholipidmembranes, said truncated tissue factor protein further characterized inthat it is soluble and that it fails to convert Factor VII to FactorVIIa yet retains catalytic activity in cleaving Factor X in the presenceof Factor VIIa.
 2. The method of claim 1, wherein said effective amountof said truncated tissue factor protein is an amount sufficient toproduce a level of from about 100 nanograms to about 50 micrograms saidtruncated tissue factor protein per milliliter of plasma and saideffective amount of said Factor VIIa is an amount sufficient to producea level of from about 20 nanograms to about 10 micrograms Factor VIIaper milliliter of plasma.
 3. The method of claim 2, wherein saidtruncated tissue factor protein is in an amount sufficient to produce alevel of from about 1 microgram to about 10 micrograms truncated tissuefactor protein per milliliter of plasma.
 4. A method according to claim3, wherein said Factor VIIa is administered to produce a level ofbetween about 40 nanograms and 4 micrograms Factor VIIa per milliliterof plasma.
 5. A method for treating a patient to control bleedingaccording to claim 1 wherein said truncated tissue factor protein andsaid Factor VIIa are administered topically to a wound site.
 6. A methodfor treating a patient to control bleeding according to claim 5 whereina sufficient amount of said truncated tissue factor protein isadministered to produce a local concentration in said wound site of fromabout 10 to about 50 micrograms truncated tissue factor protein permilliliter of plasma and a sufficient amount of said Factor VIIa isadministered to produce a local concentration in said wound site of fromabout 1 to about 10 micrograms Factor VIIa per milliliter of plasma. 7.A method for treating a patient for excessive bleeding occurring at asite, comprising the step of:sequentially administering an effectiveamount of a truncated tissue factor protein and an effective amount ofFactor VIIa to a patient to effect clotting of said patient's blood atsaid site and subsequent reduction of said excessive bleeding, saidtruncated tissue factor protein having an amino acid sequence beginningat position 1 and terminating near position 219 of SEQ ID NO:2, saidtruncated tissue factor protein differing from that of native tissuefactor protein in that transmembrane amino acids selected from themembrane spanning domain of native tissue factor protein which would besufficient to bind said truncated tissue factor protein to phospholipidmembranes are lacking from said truncated tissue factor protein so thatsaid truncated tissue factor protein does not bind to phospholipidmembranes, said truncated tissue factor protein further characterized inthat it is soluble and that it fails to convert Factor VII to FactorVIIa yet retains catalytic activity in cleaving Factor X in the presenceof Factor VIIa.
 8. The method of claim 7, wherein said effective amountof said truncated tissue factor protein is an amount sufficient toproduce a level of from about 100 nanograms to about 50 micrograms saidtruncated tissue factor protein per milliliter of plasma and saideffective amount of said factor protein VIIa is an amount sufficient toproduce a level of from about 20 nanograms to about 10 micrograms FactorVIIa per milliliter of plasma.
 9. The method of claim 8, wherein saidtruncated tissue factor protein is added to produce a level of fromabout 1 microgram to about 10 micrograms truncated tissue factor proteinper milliliter of plasma.
 10. The method of claim 9, wherein said FactorVIIa is administered to produce a level of between about 40 nanogramsand 4 micrograms Factor VIIa per milliliter of plasma.
 11. A method fortreating a patient to control bleeding according to claim 7 wherein saidtruncated tissue factor protein and said Factor VIIa are administeredtopically to a wound site.
 12. A method for treating a patient tocontrol bleeding according to claim 11 wherein a sufficient amount ofsaid truncated tissue factor protein is administered to produce a localconcentration in said wound site of from about 10 to about 50 microgramstruncated tissue factor protein per milliliter of plasma and asufficient amount of said Factor VIIa is administered to produce a localconcentration in said wound site of from about 1 to about 10 microgramsFactor VIIa per milliliter of plasma.
 13. A pharmaceutical compositionfor treating excessive bleeding comprising a truncated tissue factorprotein having an amino acid sequence beginning at position 1 andterminating near position 219 of SEQ ID NO:2, said truncated tissuefactor protein differing from that of native tissue factor protein inthat transmembrane amino acids selected from the membrane spanningdomain of native tissue factor protein which would be sufficient to bindsaid truncated tissue factor protein to phospholipid membranes arelacking from said truncated tissue factor protein protein so that saidtruncated tissue factor protein does not bind to phospholipid membranes,said truncated tissue factor protein further characterized in that issoluble and that it fails to convert Factor VII to Factor VIIa yetretains catalytic activity in cleaving Factor X in the presence ofFactor VIIa, and Factor VIIa.
 14. The composition of claim 13, furthercomprising a pharmaceutically acceptable carrier for systemicadministration.
 15. The composition of claim 13, further comprising apharmaceutically acceptable carrier for topical administration.
 16. Thecomposition of claim 15, wherein said composition comprises about 10nanograms to about 50 micrograms of said truncated tissue factor proteinper milliliter of said carrier and from about 1 to about 10 microgramsFactor VIIa per milliliter of said carrier.
 17. A pharmaceuticalcomposition according to claim 16 wherein said truncated tissue factorprotein is present in a concentration of from about 10 to about 50micrograms per milliliter of said carrier.
 18. A method for treating apatient for excessive bleeding occurring at a site, comprising the stepof:simultaneously administering an effective amount of a truncatedtissue factor protein and an effective amount of Factor VIIa to saidpatient to effect clotting of said patient's blood at said site andsubsequent reduction of said excessive bleeding, said truncated tissuefactor protein further characterized in that it does not bind tophospholipid membranes, that it is soluble and that it fails to convertFactor VII to Factor VIIa yet retains catalytic activity in cleavingFactor X in the presence of Factor VIIa.
 19. A method for treating apatient for excessive bleeding occurring at a site, comprising the stepof:sequentially administering an effective amount of a truncated tissuefactor protein and an effective amount of Factor VIIa to said patient toeffect clotting of said patient's blood at said site and subsequentreduction of said excessive bleeding, said truncated tissue factorprotein further characterized in that it does not bind to phospholipidmembranes, that it is soluble and that it fails to convert Factor VII toFactor VIIa yet retains catalytic activity in cleaving Factor X in thepresence of Factor VIIa.
 20. A pharmaceutical composition for treatingexcessive bleeding comprising a truncated tissue factor protein, saidtruncated tissue factor protein further characterized in that it doesnot bind to phospholipid membranes, that it is soluble and that it failsto convert Factor VII to Factor VIIa yet retains catalytic activity incleaving Factor X in the presence of Factor VIIa, and Factor VIIa.